Physical Properties and pH Environment of Foam Dressing Containing Eclipta prostrata Leaf Extract and Gelatin

Eclipta prostrata (E. prostrata) has several biological activities, including antibacterial and anti-inflammatory activities, that improve wound healing. It is well known that physical properties and pH environment are crucial considerations when developing wound dressings containing medicinal plant extracts in order to create an appropriate environment for wound healing. In this study, we prepared a foam dressing containing E. prostrata leaf extract and gelatin. Chemical composition was verified using Fourier-transform infrared spectroscopy (FTIR) and pore structure was obtained using scanning electron microscopy (SEM). The physical properties of the dressing, including absorption and dehydration properties, were also evaluated. The chemical properties were measured to determine the pH environment after the dressing was suspended in water. The results revealed that the E. prostrata dressings had a pore structure with an appropriate pore size (313.25 ± 76.51 µm and 383.26 ± 64.45 µm for the E. prostrata A and E. prostrata B dressings, respectively). The E. prostrata B dressings showed a higher percentage of weight increase in the first hour and a faster dehydration rate in the first 4 h. Furthermore, the E. prostrata dressings had a slightly acidic environment (5.28 ± 0.02 and 5.38 ± 0.02 for the E. prostrata A and E. prostrata B dressings at 48 h, respectively).


Introduction
Plant-based biomaterials have several benefits over synthetic materials, including cost-effectiveness, safety for humans, and their environmentally friendly composition [1]. Developing novel wound dressings containing medicinal plant extracts can improve clinical outcomes and increase plant value [2,3]. In this study, we examined Eclipta prostrata L. (E. prostrata L.) due to its pharmacological properties. E. prostrata L., commonly known as False daisy, Ink plant, Bhringraj, Bhumiraj, Aali jhar, or Nash jhar, is a herbaceous plant that belongs to the family Asteraceae [4]. It is a weed that grows in moist places such as rivers, marshes, or the edge of rice fields [4,5]. It is found in many parts of the world, including Thailand, China, India, Nepal, and Brazil [4]. It has been long used in the treatment of several diseases, including coronary heart disease, diabetes, gastrointestinal diseases, respiratory diseases, skin diseases, and wounds [5]. The leaves have various biological activities, including antibacterial, antifungal, and anti-inflammatory activities, that improve wound healing [6][7][8][9]. Therefore, a wound dressing containing E. prostrata L. extract could be used for treating infection and inflammation in wound healing. Kang et al. [10] suggest that E. prostrata L. extract is a potential treatment for inflammatory skin conditions such as atopic dermatitis. The E. prostrata L. extract improved the allergic inflammation of the skin by restoring the skin barrier dysfunction, decreasing epidermis/dermis thickness,  Table 1 shows the FTIR peak values and functional groups of the E. prostrata leaf extract and the E. prostrata A and B dressings. Figure 1 shows the FTIR spectra of the E. prostrata leaf extract. Figure 2 shows the FTIR spectra of the E. prostrata A and B dressings. After mixing gelatin and E. prostrata leaf extract and then lyophilization or freeze drying, there was an increased intensity in the functional groups (as seen in Figure 2), including amide I (1646.91 cm −1 and 1647.22 cm −1 for the E. prostrata A and B dressings, respectively), and amide II (1553.43 cm −1 and 1554.20 cm −1 for the E. prostrata A and B dressings, respectively). The amide I (mainly related to the C=O stretching vibration) and II (mainly related to the N-H bending vibration and the C-N stretching vibration) bands are associated with the presence of gelatin [35,36].  Figure 1 shows the FTIR spectra of the prostrata leaf extract. Figure 2 shows the FTIR spectra of the E. prostrata A and B dressing After mixing gelatin and E. prostrata leaf extract and then lyophilization or freeze dryin there was an increased intensity in the functional groups (as seen in Figure 2), includin amide I (1646.91 cm −1 and 1647.22 cm −1 for the E. prostrata A and B dressings, respectively and amide II (1553.43 cm −1 and 1554.20 cm −1 for the E. prostrata A and B dressings, respe tively). The amide I (mainly related to the C=O stretching vibration) and II (mainly relate to the N-H bending vibration and the C-N stretching vibration) bands are associated wit the presence of gelatin [35,36].

Morphological Properties
The surface and cross-sectional morphologies of E. prostrata dressings were observed using SEM (Figure 3). The size, shape, and distribution of pores are shown in Figure 3b. The average pore sizes were 313.25 ± 76.51 µm and 383.26 ± 64.45 µm for the E. prostrata A and B dressings, respectively (Figure 3b). The E. prostrata B dressing had more consistent porosity than E. prostrata A dressing.

Morphological Properties
The surface and cross-sectional morphologies of E. prostrata dressings were observed using SEM (Figure 3). The size, shape, and distribution of pores are shown in Figure 3b. The average pore sizes were 313.25 ± 76.51 µm and 383.26 ± 64.45 µm for the E. prostrata A and B dressings, respectively ( Figure 3b). The E. prostrata B dressing had more consistent porosity than E. prostrata A dressing.

Morphological Properties
The surface and cross-sectional morphologies of E. prostrata dressings were observed using SEM (Figure 3). The size, shape, and distribution of pores are shown in Figure 3b. The average pore sizes were 313.25 ± 76.51 µm and 383.26 ± 64.45 µm for the E. prostrata A and B dressings, respectively (Figure 3b). The E. prostrata B dressing had more consistent porosity than E. prostrata A dressing.

Absorption Properties
The absorption properties of the dressing determine how well it can manage wound exudate and promote wound healing. The percentage of weight increase of the E. prostrata dressing for different periods is shown in Figure 4. The E. prostrata A dressing showed a lower percentage of weight increase than the E. prostrata B dressing. The higher E. prostrata leaf extract promotes the higher absorption capacity.

Dehydration Properties
The dehydration rate of the E. prostrata dressing for different periods is presented in Figure 5. The E. prostrata B dressing showed a higher dehydration rate in the first 4 h than the E. prostrata A dressing. The higher E. prostrata leaf extract promotes the higher dehydration rate.

Absorption Properties
The absorption properties of the dressing determine how well it can manage wound exudate and promote wound healing. The percentage of weight increase of the E. prostrata dressing for different periods is shown in Figure 4. The E. prostrata A dressing showed a lower percentage of weight increase than the E. prostrata B dressing. The higher E. prostrata leaf extract promotes the higher absorption capacity.

Absorption Properties
The absorption properties of the dressing determine how well it can manage wo exudate and promote wound healing. The percentage of weight increase of the E. pro dressing for different periods is shown in Figure 4. The E. prostrata A dressing show lower percentage of weight increase than the E. prostrata B dressing. The higher E. pro leaf extract promotes the higher absorption capacity.

Dehydration Properties
The dehydration rate of the E. prostrata dressing for different periods is present Figure 5. The E. prostrata B dressing showed a higher dehydration rate in the first 4 h

Dehydration Properties
The dehydration rate of the E. prostrata dressing for different periods is presented in Figure 5. The E. prostrata B dressing showed a higher dehydration rate in the first 4 h than the E. prostrata A dressing. The higher E. prostrata leaf extract promotes the higher dehydration rate.  Figure 6 demonstrates the pH of deionized water after the E. prostrata dressings were submerged in it. The pH of the deionized water with the submerged E. prostrata A dressing decreased from 7.55 ± 0.16 to 5.28 ± 0.02. In the same way, the pH of deionized water with the submerged E. prostrata B dressing decreased from 7.69 ± 0.24 to 5.38 ± 0.02.  2.6. pH Measurement Figure 6 demonstrates the pH of deionized water after the E. prostrata dressings were submerged in it. The pH of the deionized water with the submerged E. prostrata A dressing decreased from 7.55 ± 0.16 to 5.28 ± 0.02. In the same way, the pH of deionized water with the submerged E. prostrata B dressing decreased from 7.69 ± 0.24 to 5.38 ± 0.02.  Figure 6 demonstrates the pH of deionized water after the E. prostrata dressings were submerged in it. The pH of the deionized water with the submerged E. prostrata A dressing decreased from 7.55 ± 0.16 to 5.28 ± 0.02. In the same way, the pH of deionized water with the submerged E. prostrata B dressing decreased from 7.69 ± 0.24 to 5.38 ± 0.02.

Dispersion Characteristics
The dispersion characteristics of the E. prostrata dressings are shown in Figures 7 and  8. The E. prostrata A and B dressings did not change much from their original structure after they were immersed in pseudo-wound exudate for 60 s at 100 revolutions per minute

Dispersion Characteristics
The dispersion characteristics of the E. prostrata dressings are shown in Figures 7 and 8. The E. prostrata A and B dressings did not change much from their original structure after they were immersed in pseudo-wound exudate for 60 s at 100 revolutions per minute ( Figure 7). However, the spectra of the pseudo-wound exudate after the E. prostrata A and B dressings were submerged in it were not similar to those of the pseudo-wound exudate ( Figure 8).

Discussion
E. prostrata leaf extract has been studied for its potential wound-healing benefits, including its antimicrobial and anti-inflammatory properties [6][7][8][9]. Prior studies examining the development of wound healing products containing E. prostrata focus only on ointment and hydrogel formulations [11,12]. No studies have developed wound dressings containing E. prostrata leaf extract in sheet form or foam dressings. Developing wound dressing in sheet form has several advantages, including preventing trauma, minimizing external contamination, absorbing exudate, and keeping a wound in an optimally moist environment [16].
In this study, a foam dressing containing E. prostrata extract and gelatin was developed to evaluate the physical properties and pH wound environment. The E. prostrata dressings were soft and flexible. These properties help to maintain a moist wound environment, reduce the risk of maceration, and allow use in the movement areas, such as the knee or elbow [37]. The E. prostrata A dressing was thicker than the E. prostrata B dressing. This is explained as the high protein content in bovine gelatin increased the polymer matrix's solids content. Hence, the increase in gelatin or protein concentration has induced an increase in the thickness of the foam dressing [38]. However, the thickness was unrelated to the absorption and dehydration properties, as shown in Figures 4 and 5.
The FTIR spectra are used to identify the functional groups present in the E. prostrata dressing, as compared to the E. prostrata leaf extract. It was found that the FTIR spectra of E. prostrata dressing had an increased intensity in the functional groups, including amide I and II (Figure 2). The amide I and II bands in the FTIR spectra are commonly used to identify the presence of gelatin [35,36]. The amide I band in FTIR spectra is a strong absorption peak that corresponds to the stretching vibration of the C=O bond in the peptide backbone [39]. This band of gelatin appears in the region of 1600-1700 cm −1 [35]. As shown in Figure 2, the amide I band was around 1646-1648 cm −1 , indicating the presence of a predominantly random coil structure [39]. The amide II band in the FTIR spectra I also provide information on the vibrational bands of the protein backbone [39]. This band corresponds to the bending vibration of the N-H bond (40-60% of the potential energy) and the stretching vibration of the C-N bond (18-40%) in the protein backbone [39]. In the case of gelatin, the amide II band appears in the region of 1565-1520 cm −1 [35]. As shown in Figure 2, the amide II band was around 1553-1555 cm −1 . The amide II band is often used in combination with the amide I band to confirm the presence of gelatin. Therefore, it indicates that our process to develop an E. prostrata dressing did not affect the structural property of gelatin. Gelatin could provide a porous structure and produce biodegradable and biocompatible material [31,34].
The resulting SEM image provides information about the morphology or porous structure of the E. prostrata dressing (Figure 3). A porous structure is crucial in wound healing because it allows cell migration and proliferation [28,40]. When a wound occurs, the first phase of wound healing is hemostasis, with vascular constriction, platelet aggregation, degranulation, and fibrin clot formation [29]. Hemostasis helps to stop bleeding, and inflammatory cells (neutrophils, monocytes, macrophages, and lymphocytes) migrate into the wound, triggering the inflammatory response (also known as the "inflammatory phase") [29]. The next phase is proliferation, with re-epithelialization, angiogenesis, collagen synthesis, and extracellular matrix (ECM) formation; this generally overlaps with the inflammatory phase [29]. The porous structure supports this phase. Fibroblasts and endothelial cells need to migrate into the wound bed in order to proliferate and form granulation tissue at the site of injury [29]. A porous structure allows for these cells to migrate into the wound bed, promoting efficient wound healing. Following cell proliferation, the final phase is remodeling (collagen remodeling and vascular maturation) and regression [29]. A previous study by Murphy et al. [41] showed that a mean pore size of 325 µm facilitated the highest cell attachment and proliferation when compared with pores in the 85-190 µm range. As seen in Figure 3b, our SEM images of the cross-section show an average pore size of around 300 µm. This ensured that the E. prostrata A and B dressings had an appropriate pore size for efficient wound healing. Nevertheless, the E. prostrata B dressing had a more consistent porosity than the E. prostrata A dressing. The effect of this difference in porosity between the E. prostrata A and B dressings could result in differences in absorption ability.
We developed the E. prostrata dressing, which was designed with a porous structure, in order to increase absorption ability. In this study, absorption ability was obtained by using pseudo-wound exudate. The E. prostrata B dressing exhibited a stronger absorption ability than the E. prostrata A dressing (Figure 4). The absorption ability of the E. prostrata B dressing was derived from a higher-density porous structure (Figure 3b). The ideal wound dressing must absorb excess wound exudate and provide a moist environment [18,21,22,42]. Wound exudate or wound drainage is the fluid that discharges from a wound during the healing process [43]. The mechanism of exudate formation is usually due to inflammation or infection [43]. The amount of exudate produced can vary depending on the type and severity of the wound. A moist wound environment is necessary for the woundhealing process to occur effectively. An optimal moisture level enhances cell migration and proliferation, reduces pain and discomfort, and reduces infection rates [22,23]. Macerated peri-wound skin can lead to an increased risk of infection, whereas desiccated peri-wound skin can lead to decreased epithelial migration and cell death [22,44]. Therefore, the selection of absorbent wound dressing depends on the amount of exudate to prevent both maceration and desiccation. Moreover, a moist environment promotes autolysis or breakdown of necrotic tissue, called autolytic debridement [23,45]. In our previous work [20], the commercial hydrocolloid dressing and hydrocolloid with foam layer dressing had the lowest absorption capacity. Therefore it is an appropriate dressing for wounds with a low amount of exudate. In this study, both the E. prostrata A and B dressings had absorption characteristics similar to commercial hydrocolloid dressings and hydrocolloid with foam layer dressings [20]. These absorption characteristics meant that both the E. prostrata A and B dressings could be chosen for wounds with low exudate.
Dehydration rate is also essential to control the moisture balance of the wound and enhance wound healing as a result of water-retaining properties [22,46]. This can be achieved through the use of appropriate wound dressings that are designed to manage moisture levels and prevent dehydration. In addition, the selection of wound dressing also depends on the amount of exudate produced by the wound. The E. prostrata B dressing showed a higher dehydration rate than the E. prostrata A dressing. This can be explained by the higher-density porous structure of the E. prostrata B dressing (Figure 3b). Therefore, the E. prostrata B dressing can dehydrate exudate to rapidly create a moist wound-healing environment.
The pH of the wound environment is an essential factor for wound healing. The pH of healthy human skin is in the range of 5.4 to 5.9, which is slightly acidic. [47]. Propionibacterium is commonly found on human skin. Propionibacterium grow well at pH 6.00-6.50 [48]. Staphylococcus aureus is a pyogenic bacterium [49]. S. aureus prefers a neutral pH environment for optimal growth and survival [50]. Thus, an acidic environment is not favorable for harmful bacterial growth. In addition, the pH environment of chronic wounds exists at a range of 7.15 to 8.90; this is alkaline and chronic wounds are characterized by excessive protease activity [51][52][53][54][55]. Sim et al. found that faster recovery of wounded tissues was observed in wounds treated by pH 4 buffers when compared to pH 6 buffers [25]. A previous study by Leveen et al. showed that a slightly acidic environment significantly inhibits protease activity and may potentially enhance the healing of cutaneous wounds [56]. Previous studies reported fibroblast proliferation and migration behaviors associated with the acidic environment [57,58]. It means rapid wound healing occurs in an acidic environment [25,54]. We found that the E. prostrata A and B dressings showed similar pH decreases continuously over the period. Our E. prostrata dressings tended to create a slightly acidic environment. Hence, it was supposed that the E. prostrata A and B dressings would not interfere with the wound healing process.
The dispersion of the wound dressing refers to how well the dressing covers the wound surface. In this study, the spectra of the pseudo-wound exudate after the E. prostrata A and B dressings were submerged in it were not similar to those of the pseudo-wound exudate (Figure 8). In our previous study, commercial alginate dressings had the spectra of the pseudo-wound exudate after the dressings were submerged was also not similar to those of the pseudo-wound exudate [20]. Nevertheless, after interacting with the pseudowound exudate, the Eclipta prostrata A and B dressings did not change substantially from their original structure (Figure 7). It means that the E. prostrata dressing will not be difficult to remove. According to the spectra of the pseudo-wound exudate after the dressings were submerged, our E. prostrata dressings have an immediate-release formulation. The E. prostrata dressing should be further modified for controlled release applications by crosslinking techniques with a crosslinker, such as glutaraldehyde [59,60].

Preparation of Foam Dressing Containing E. prostrata Extract and Gelatin
The gelatin solution (10% w/v) was prepared by dissolving the gelatin in deionized water at 40 • C and stirring continuously for 1 h. Then, the E. prostrata dressing was prepared by mixing gelatin solution and E. prostrata leaf extract, as shown in Table 2. The mixture was stirred for 1 h to obtain a homogeneous solution. After stirring, the solution was sonicated to eliminate air bubbles and then poured into plastic plates. The plastic plates were placed into a freezer at −80 • C and frozen for 24 h. The frozen solution was then lyophilized in a freeze-dryer (SHM 021) for 48 h to become a sponge. Finally, the sponge (E. prostrata dressing) was slowly removed from the plastic plate. In order to prevent contamination, the E. prostrata dressings were then stored inside an airtight container. The composition with E. prostrata leaf extract of more than 40% could not prepare foam dressing.

Thickness Test
After lyophilization, a thickness test was performed using the Mitutoyo Dial Thickness Gauge, which provided an accuracy of 0.001 mm. Thickness was measured at five different positions (one in the center and four in the middle of each side).

Fourier Transform Infrared Spectroscopy (FTIR)
The E. prostrata leaf extracts and the E. prostrata dressings were recorded on a spectrum 100 FTIR Spectrometer (PerkinElmer Inc., Waltham, MA, USA) FTIR spectra were recorded from 500 to 4000 cm −1 .

Morphological Properties
At a voltage of 10 kV, the E. prostrata dressings were examined using a Scanning Electron Microscope (SEM, JSM-IT300 JEOL). SEM with an Energy Dispersive X-ray Spectrometer (EDS) was used to analyze the dressings with the surface (500×) and cross-sectional (60×) images. The E. prostrata dressing was first prepared by attaching it to the aluminium stubs and then coating it with gold. This process helps to improve the conductivity of the dressing, allowing for better imaging results. The pore sizes were measured using the Image J ® software (National Institutes of Health, Bethesda, MA, USA).

Absorption Properties
The absorption properties of the E. prostrata dressing were examined using BS EN 13726-1: 2002, Part 1: the aspects of absorbency, Section 3.2: free swell absorptive capacities with slight modifications [61]. The E. prostrata dressing (2 cm × 2 cm) was prepared and weighed. A test solution (8.298 g of NaCl (0.142 mol/L) and 0.367 g of CaCl 2 2H 2 O (0.0025 mol/L)) was added to one liter of deionized water, representing a pseudo-wound exudate. The E. prostrata dressing was immersed in the test solution and then incubated at 37 • C. At different periods, the dressing was removed and weighed.

Dehydration Properties
The E. prostrata dressing (2 cm × 2 cm) was prepared and weighed. The E. prostrata dressing was immersed in the test solution (pseudo-wound exudate) for 30 min. Then, the dressing was removed, weighed, and incubated at 37 • C. At different periods, the dressing was weighed [62].

pH Measurement
The E. prostrata dressing (2 cm × 2 cm) was suspended in deionized water at a ratio of 1:25 (w/v). At different periods, the deionized water was measured using a pH meter (pH 700) [62].

Dispersion Characteristics
The dispersion characteristics of the E. prostrata dressing were examined using BS EN 137262: 2002, Part 1: the aspects of absorbency, Section 3.6: dispersion characteristics with slight modifications [63]. The E. prostrata dressing (2 cm × 2 cm) was prepared and immersed in the test solution and shaken for 60 s at 100 revolutions per minute. Then, the absorbance of the collected test solution was measured using a UV-spectrophotometer (UV-2501PC) by scanning between a wavelength of 200 and 450 nm.

Statistical Analysis
The experiments were performed in triplicate and represented in a mean ± standard deviation.

Conclusions
This study is the first development of wound dressing sheets containing E. prostrata leaf extract and gelatin. Our study investigated the physical properties and pH pseudowound environment of the E. prostrata dressing. Both the E. prostrata A and B dressings had an appropriate pore size (313.25 ± 76.51 µm and 383.26 ± 64.45 µm, respectively) for cell migration and proliferation in the wound healing process. Greater E. prostrata leaf extract produces a higher-density porous structure of foam dressing, resulting in a higher absorption capacity and faster dehydration rate. The E. prostrata dressings are designed for the low level of exudate due to their absorption capacity. In addition, the E. prostrata dressings make the environment slightly acidic. Therefore, it was supposed that our E. prostrata dressings would not provide favorable conditions for bacterial growth. Our results provide the wound dressing profiles that are essential for the decision of physicians to select the appropriate wound dressing according to the amount of exudate. Further experimental studies should focus on release patterns, pharmacological properties, such as antibacterial and anti-inflammatory activities, and wound healing assays.